The invention provides a thin-well microplate having an array of sample wells and a combination of specific physical and material properties required for use with automated equipment, such as robotic handling equipment, to withstand conditions of thermal cycling procedures and provide optimal thermal transfer and biological properties. The invention also provides methods of constructing the thin-well microplate as a unitary plate, employing ideal materials of construction to impart and optimize specific physical and material properties of the thin-well microplate.
Various biological research and clinical diagnostic procedures and techniques require or are facilitated by an array of wells or tubes in which multiple samples are disposed for qualitative and quantitative assays or for sample storage and retrieval. Prior art devices that provide an array of wells or tubes capable of containing small sample volumes include microtitration plates that are commonly known as multi-well plates.
Multi-well plates have open-top wells, cups or recesses capable of containing small volumes of typically aqueous samples ranging from fractions of a microliter to hundreds of microliters. Multi-well plates also typically include sample well arrays totaling 96 sample wells that are arranged in an array of 8 by 12 sample wells and have center-to-center well spacing of 9 mm, such as the multi-well plate disclosed in U.S. Pat. No. 3,356,462. Sample well arrays also include arrays of 384 wells arranged in 16 by 24 array with a reduced center-to-center well spacing of 4.5 mm. Well arrays are not limited to any particular number of wells nor to any specific array pattern. For example, U.S. Pat. No. 5,910,287 discloses a multi-well plate comprising a well array of more than 864 wells.
Research techniques that use multi-well plates include, but are not limited to, quantitative binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbant assay (ELISA), combinatorial chemistry, cell-based assays, thermal cycle DNA sequencing and polymerase chain reaction (PCR), both of which amplify a specific DNA sequence using a series of thermal cycles. Each of these techniques makes specific demands on the physical and material properties and surface characteristics of the sample wells. For instance, RIA and ELISA require surfaces with high protein binding; combinatorial chemistry requires great chemical and thermal resistance; cell-based assays require surfaces compatible with sterilization and cell attachment, as well as good transparency; and thermal cycling requires low protein and DNA binding, good thermal conductivity, and moderate thermal resistance.
Different uses of multi-well plates make different demands on the overall form and structure of the multi-well plate. The compatibility of plates with automated equipment is perhaps one of the most stringent constraints on the form and structure of plates. Many laboratories automate various steps or phases of procedures, such as depositing and removing small quantities of reaction mixture from sample wells, often 5 xcexcl or less, using automated dispensing/aspiration systems. Furthermore, plate handling equipment is often used to help facilitate the automation of such procedures. Accordingly, it is desirable to use a multi-well plate that is conducive to use with robotic equipment and can withstand robotic gripping and manipulation.
Efforts to standardize the features which permit successful deployment of multi-well plates in robotic handling and liquid handling instruments have been recommended (Society of Biomolecular Screening Recommended Microplate Specifications http://sbsonline.com/sbs070.htm), and significant effort has been made to achieve a common geometry of key elements of multi-well plate design, including footprint (defined as length and width at the base plane), well location with respect to the exterior of the footprint, and overall flatness as well as rigidity in the robotic gripping area.
Multi-well plates used in thermal cycling procedures form a sub-set of multi-well plates and may be referred to as thin-well microplates. Use in thermal cycling places additional material and structural requirements on the thin-well microplates. Typically, multi-well plates are not exposed to high temperatures or to rapid temperature cycling. Thin-well microplates are designed to accommodate the stringent requirements of thermal cycling. For example, thin-well microplates typically have design adaptations that are intended to improve thermal transfer to samples contained within sample wells. Sample wells of thin-well microplates have thin walls typically on the order of less than or equal to 0.015 inch (0.38 mm). Sample wells typically are conical shaped to allow wells to nest into corresponding conical shapes of heating/cooling blocks of thermal cyclers. The nesting feature of sample wells helps to increase surface area of thin-well microplates while in contact with heating/cooling blocks and, thus, helps to facilitate heating and cooling of samples.
As described above with respect to standard multi-well plate applications, many laboratories utilizing thin-well microplates now automate procedures performed prior to and subsequent to thermal cycling and employ robotic equipment to facilitate such automation. To ensure reliable and accurate use with robotic instruments, the subset of thin-well microplates must also possess general physical and material properties which facilitate robotic handling as well as enable thin-well microplates to retain their dimensional stability and integrity when exposed to high temperatures of thermal cycling.
Thin-well microplates require a specific combination of physical and material properties for optimal robotic manipulation, liquid handling, and thermal cycling. These properties consist of rigidity, strength and straightness required for robotic plate manipulation; flatness of sample well arrays required for accurate and reliable liquid sample handling; physical and dimensional stability and integrity during and following exposure to temperatures approaching 100xc2x0 C.; and thin-walled sample wells required for optimal thermal transfer to samples. These various properties tend to be contradictory. For instance polymers offering improved rigidity and/or stability typically do not possess the material properties required to be biologically compatible and/or to form thin-walled sample tubes. Existing thin-well microplates are not constructed to impart all of these properties.
The typical manufacturing process for multi-well plates is polymer injection molding due to the economy of such processes. To insure multi-well plates consistently adhere to specifications for rigidity and flatness, manufacturers of prior art multi-well plates employ one or both of two design options, namely incorporating structural features with multi-well plates and using suitable and economical polymers to construct multi-well plates.
The first option of incorporating structural features with multi-well plates includes incorporating ribs with the undersides of multi-well plates to reinforce flatness and rigidity. However, such structural features cannot be incorporated with thin-well microplates used in thermal cycling procedures. Such structural features would not allow samples wells to nest in wells of thermal cycler blocks and, therefore, would prevent effective coupling with block wells resulting in less effective thermal transfer to samples contained within sample wells.
The second option to enhance rigidity and flatness of multi-well plates includes using suitable, economical polymers that impart rigidity and flatness to the plates. Simultaneously the selected polymer must also meet the physical and material property requirements of thin-well microplate sample wells in order for such sample wells to correctly function during thermal cycling. Many prior art multi-well plates are constructed of polystyrene or polycarbonate. Polystyrene and polycarbonate resins exhibit mold-flow properties that are unsuitable for forming the thin walls of sample wells that are required of thin-well microplates. Molded polystyrene softens or melts when exposed to temperatures routinely used for thermal cycling procedures. Therefore, such polymer resins are not suitable for construction of thin-well microplates for thermal cycling procedures.
Prior art thin-well microplates are also typically manufactured by injection molding processes, wherein the entire microplate is constructed in a single manufacturing operation of a single material, typically polypropylene or polyolefin. Construction of thin-well microplates by injection molding polypropylene is desirable because the flow properties of molten polypropylene allow consistent molding of a sample well with a wall that is sufficiently thin to promote optimal heat transfer when the sample well array is mounted on a thermal cycler block. In addition, polypropylene does not soften or melt when exposed to high temperatures of thermal cycling. However, prior art thin-well microplates constructed of a single polymer resin, such as polypropylene and polyolefin, in a single manufacturing operation possess inherent internal stresses found in molded parts with complex features and exhibit thick and thin cross sectional portions throughout the body of the plate. Internal stresses result from differences in cooling rate of thick and thin portions of the plate body after a molding process is complete. In addition, further distortions, such as warping and shrinkage due to internal stresses, can result when thin-well microplates are exposed to conditions of thermal cycling procedures. Also, the resultant dimensional variations in flatness and footprint size can lead to unreliable sample loading and sample recovery by automated equipment.
Alternative prior art manufacturing methods include thermoforming thin-well multi-well plates from polycarbonate sheet material, such as product number 9332 available from Corning of Corning, New York and product number CON-9601 from MJ Research, Inc. of Waltham, Mass. Thin-well microplates manufactured by thermoforming polycarbonate, however, do not provide the rigidity and dimensional precision required of thin-well microplates for use with robotic equipment, nor the dimensional precision required for accurate liquid dispensing and aspiration by automated sample handling equipment.
Prior art thin-well polycarbonate microplates that have been promoted for robotic applications continue to exhibit dimensional variations associated with thin-well polypropylene microplates. Such thin-well polypropylene microplates thus limit the reliability and precision with which such microplates may be used with robotic equipment. In addition, such thin-well polypropylene microplates require external rigid adaptors to restore dimensional precision, such as Microseal 384 Plate Positioner, product number ADR-3841 available from MJ Research, Inc. of Waltham, Mass. Attempts to increase thin-well microplate rigidity by increasing overall thickness of molded parts of such microplates have resulted in an undesirable increase in the thickness of sample well walls, such as UNI PCR 96-well plate available from Polyfiltronics, Inc. of Rockland, Mass., wherein the average sample well wall thickness is greater than or equal to 0.020 inches (0.5 mm).
Using currently available manufacturing methods, the requirements for robotic-compatible thin-well microplates are in direct conflict with the requirements for thin-well microplates for use in thermal cycling procedures. One known method of addressing this problem is to utilize a tray of a first material with sample wells separately created from a second material. Such microplates are commercially available are under the names of xe2x80x9cOmni-Tube Platexe2x80x9d and xe2x80x9cThermo-Tube Platexe2x80x9d, available from ABgene Ltd. of Surrey, UK. Both products consist of a tray, with overall dimensions approximating those of a multi-well plate, having an array of holes into which separately manufactured tubes or strips of tubes are loosely inserted. Because of the assembly required, these products do not offer the convenience of a single, unitary plate provided by a thin-well microplate. The high throughput nature of automated microplate processes inherently requires that manual intervention be minimized. Such a high throughput nature also precludes any preparatory or assembling steps, such as assembly of a sample vessel or microplate from various component parts. Further, the geometry and loosely fitting nature of these products does not lend these products to use with high-precision robotic equipment and automated dispensing equipment.
Therefore, it is desirable to provide a thin-well microplate as a single, unitary plate that is compatible for use with high-precision robotic handling equipment in automated procedures. A thin-well microplate that possesses the physical and material properties to maintain dimensional stability and integrity during robotic handling under the high temperature conditions of the thermal cycling procedures while also possessing properties that are conducive to thermal cycling reactions is also highly desirable.
Embodiments of the invention are directed to a thin-well microplate for use in research procedures and diagnostic techniques and to methods of manufacturing same. The thin-well microplate of the invention comprises a unitary plate of two separate components including a skirt and frame portion and a well and deck portion having a plurality of sample wells. Each portion is constructed as a separate component of a suitable material that is selected for the specific physical and material properties such material imparts to each component. The skirt and frame portion and the well and deck portion are joined to form the unitary plate. The combination of physical and material properties provided by the skirt and frame portion and the well and deck portion includes, although not limited to, thin-walled sample wells for adequate thermal transfer and physical stability to withstand high temperature conditions. The combination of physical and material properties provided by the skirt and frame portion and the well and deck portion optimizes the performance of the thin-well microplate with automated equipment in thermal cycling procedures.
In a first embodiment of the invention, a thin-well microplate includes a skirt and frame portion with a top surface having an plurality of holes arranged in a first array pattern and a well and deck portion joined to the top surface of the skirt and frame portion to form a unitary plate. The well and deck portion includes a plurality of sample wells integral with the deck and portion and arranged in the first array pattern such that the sample wells extend through the plurality of holes of the skirt and frame portion when the well and deck portion is joined with the skirt and frame portion to form the unitary plate. The skirt and frame portion is constructed of a first material that imparts rigidity to the skirt and frame portion to allow the thin-well microplate to be used with automated equipment. The well and deck portion is constructed of a second material that forms sample wells with thin walls of consistent thickness to allow adequate thermal transfer to the sample wells. The second material of construction further allows the thin-well microplate to be used with optical detection equipment due to sufficient opacity provided by the second material to the sample wells.
The unitary plate of the first embodiment includes the skirt and frame portion and the well and deck portion formed as separate components and then permanently joined to form the unitary plate. In another version of the first embodiment, the well and deck portion is formed integral with the top surface of the skirt and frame portion to form the unitary plate.
The skirt and frame portion includes four walls forming a bottom opposite the top surface, wherein the bottom has a length and width slightly larger than the length and width of the top surface. The skirt and frame portion further includes at least one indentation in each wall to allow engagement of automated equipment with the thin-well microplate.
The well and deck portion further includes a raised rim around an opening of each sample well that is contiguous with an upper surface of the well and deck portion. The raised rim forms grooves in the well and deck portion between adjacent sample wells to prevent contamination between sample wells.
In another embodiment of the invention, the well and deck portion includes an upper surface having a plurality of interconnecting links with individual links joining adjacent sample wells to form a meshwork of interconnecting links and sample wells. As described above, the well and deck portion including the meshwork of interconnecting links and sample wells may be formed as a separate component of the skirt and frame portion and then permanently joined to the skirt and frame portion to form the unitary plate. Alternatively, in a version of this embodiment, the meshwork may be formed integral with the top surface of the skirt and frame portion.
In still another embodiment of the invention, the thin-well microplate includes a skirt and frame portion, constructed of a first material, having a top surface with a plurality of holes arranged in a first array pattern, and walls of equal depth extending from the top surface. The skirt and frame portion further includes a plurality of sample wells, constructed of a second material, and arranged in the first pattern such that the sample wells extend through the plurality of holes in the top surface of the skirt and frame portion. In a version of this embodiment, the thin-well microplate includes a plurality of interconnecting links with individual links joining adjacent sample wells.
In the first embodiment, the first material used to construct the skirt and frame portion is, although not limited to, a polymer resin or a filled polymer resin. The filled polymer resin is capable of withstanding a temperature of at least 100xc2x0 C. which allows the thin-well microplate to be used in thermal cycling procedures in which high temperatures are used. The skirt and frame portion in one version of the first embodiment is constructed of glass-filled polypropylene which imparts sufficient rigidity to the skirt and frame portion to allow the thin-well microplate to be used with automated equipment.
The second material used to construct the well and deck portion of the first embodiment is, although not limited to, a polymer resin or an unfilled polymer resin. The unfilled polymer resin is capable of withstanding a temperature of at least 100xc2x0 C., which similarly allows the thin-well microplate to be used in high temperature thermal cycling procedures. However, the unfilled polymer resin not only withstands high temperature conditions of thermal cycling, but forms sample wells with thin walls of consistent thickness. In one version of this embodiment, the well and deck portion is constructed of an unfilled polypropylene which forms sample wells with thin walls to allow adequate thermal transfer to sample wells during thermal cycling procedures, and also provides sufficient opacity to the sample wells to allow use of optical detection equipment with the thin-well microplate.
The invention is also directed to methods of construction of the thin-well microplate. Methods of construction include in one embodiment a first method of construction wherein the thin-well microplate is formed as a unitary plate in a single molding process comprising two steps. The first method of construction includes providing a first material that is conducive to the molding process, and molding an insert of the first material in a first step, wherein the insert includes a plurality of holes formed in a top surface of the insert. The first method of construction further includes providing a second material that is conducive to the molding process, positioning the insert to receive the second material and applying the second material to the insert in a second step, wherein an over-mold is molded having a planar deck integrally formed with a top surface of the insert and a plurality of sample wells integrally formed with the top surface of the insert and the plurality of holes to produce the unitary plate.
In a version of this embodiment, the molding process is an injection molding process including the first step as a first injection molding of the first material and the second step as a second injection molding of the second material. In other versions of this embodiment, the first and second materials are polymer resins, or, alternatively, the first material is a glass-filled polypropylene and the second material is an unfilled polypropylene.
Another embodiment of the methods of construction includes a second method of construction, wherein the thin-well microplate is formed as a unitary plate in two separate manufacturing processes. The second method of construction includes providing a first material that is conducive to a first manufacturing process, forming a skirt and frame portion of the first material by the first manufacturing process, wherein the skirt and frame portion includes a plurality of holes formed in a top surface of the skirt and frame portion. The second method of construction further includes providing a second material that is conducive to a second manufacturing process and forming a well and deck portion of the second material by the second manufacturing process, wherein the well and deck portion includes a plurality of sample wells formed in a top planar deck of the well and deck portion that are sized for insertion into the plurality of holes of the skirt and frame portion. According to the second method of construction, the skirt and frame portion and the well and deck portion are joined after their separate manufacture such that the plurality of sample wells is disposed in the plurality of holes. The well and deck portion is permanently adhered to the top surface of the skirt and frame portion to produce the unitary plate.
In a version of the second method of construction of the thin-well microplate, the first and second manufacturing processes are not only separate processes, but different methods of construction. The first and the second manufacturing processes may be different methods of molding, for instance, wherein the first manufacturing process is a convention molding process and the second manufacturing process is an injection molding process. Alternatively, in another version of the second embodiment, the first and the second manufacturing processes are similar methods of manufacturing.
The second method of construction of the thin-well microplate allows the first and second manufacturing processes to each employ different materials of construction. Accordingly, another version of this embodiment includes, for instance, the first manufacturing process employing a glass-filled polypropylene to form the skirt and frame portion and the second manufacturing process employing an unfilled polypropylene to form the well and deck portion, thereby forming a unitary plate constructed of two different materials. Still another version of this embodiment of constructing the thin-well microplate in two separate manufacturing processes includes constructing the skirt and frame portion in the first manufacturing process of the first material that is a material other than a polymer resin, such as aluminum sheet stock, and constructing the well and deck portion in the second manufacturing process of the second material including an unfilled polypropylene.
Although the second method of construction of the thin-well microplate includes using different materials in each of two different or similar, but separate, processes, to construct the skirt and frame portion and the well and deck portions as separate components, the skirt and frame portion and the well and deck portion are thereafter permanently joined by adhering steps that may include, for instance, ultrasonic or thermal welding, to form the unitary plate of the invention.